Cellular functions can be broadly divided into two general categories: proliferation (reproduction) and differentiation (specialization of function). According to present theory, the proliferative function is continuously present in the normal cell, and is dominated in the mature cell by the differentiative function, which thus acts as an integrative force to regulate both differentiative and proliferative functions in the mature cell. A failure in the biochemical mechanisms upon which the cell is dependent for control of cell differentiative and proliferative functions thus has important implications, as disruption of normal differentiative and proliferative controls may result in both abnormal cellular function and abnormal cellular growth regulation. Thus, improperly enhanced cellular proliferation, particularly when coupled to impaired cellular differentiation may be the basis for neoplasia. Similarly, the well-known phenomenon of cellular senescence involves a failure of proliferation of terminally differentiated cells after a defined number of cellular generations.
This theory presents the possibility of regulating cell behavior by the use of agents which independently influence cell growth (proliferation) or differentiation. The identification of such agents is of particular interest, for example, because such agents have the potential to induce expression of cellular genes which code for cellular functions and structures characteristic of the normal mature cell type, thereby, e.g., normalizing the cell type, or leading to increased production of cellular products of commercial interest. Similarly, the identification of agents which appropriately enhance cellular proliferation may increase the normally limited lifespan of such cells (and the animals which contain them).
Such phenomena are explored in Mann, P. L., "In vitro Differentiation of Human Peripheral Blood Leukocytes: Considerations for Monoclonal Antibodies" in Radioimmunology and Radiotherapy, 121-141, Burchiel et al., eds., Elsevier (1983), with particular reference to the induction of functionally differentiating cells with limited proliferative capacity. In vitro induction of differentiation in leukocytes with fractionated pokeweed mitogen, anti-immunoglobulin and lipopolysaccharide, followed by exposure to antigen, produced particularly good antibody responses over an extended culture life. Particularly noted was the dual role of pokeweed mitogen as a function of concentration. At certain concentrations of the mitogen, termed "proliferative" concentrations, leukocyte antibody production was stimulated in vitro by inducing proliferation of the cells; however, under this stimulation, the cells rapidly age and prematurely die. In contrast, at other, "differentiative" concentrations of the same mitogen, long-term cultures of differentiated cells producing antigen-specific immunoglobulins at stabilized levels were obtained.
The recognition of proliferative/differentiative cellular phenomena has led to the postulation of a variety of mechanisms by which cellular function is controlled in vivo and by which cellular function may be manipulated.
It has been hypothesized (e.g., Mann, P. L., in Intl. Rev. Cyto. 112, 67-96 (1988)) that there are biomolecules which control and integrate cellular differentiation. The hypothesis suggests that there are various levels of differentiation control related to evolutionary development. Primitive differentiation mechanisms were required at the early stages of cellular evolution to define the cellular boundary, inside versus outside (the modern concept of self/non-self is based on this primitive beginning). These mechanisms were focused at the boundary of the cell, that is the cell surface. Primitive cellular differentiation mechanisms thus are those which relate to the basal differentiation control of cell phenotype and interaction.
The assumption contends that complexity of control developed from, and was integrated into, these primitive mechanisms as a result of increased functional complexity. In addition to the simple primitive regulatory/recognition, a myriad of highly specific and sophisticated mechanisms have been developed as evolution has continued to fine-tune cellular communication and adapt to multi-cell, and organismic existence, and therefore, those primitive mechanisms are common to all cells, and are non-cell-lineage specific. Two models have been used to study these events and control: cellular senescence, a normal growth controlled cellular population which loses the ability to replenish itself; and a neoplastic cell population which does not express any growth control, the antithesis of cellular senescence.
Cellular senescence according to Bell et al. (Science 202, 1158-1163 (1978)) is a terminal differentiation condition, presumably one in which the population is expressing its final, natural differentiation state. According to the present hypothesis, this cell population is simply no longer able to integrate growth control information in an appropriate way at the cell surface. The growth transformed cell model, on the other hand shows aberrant growth regulation and no apparent terminal phenotype.
The cell surface oligosaccharides of both normal and growth transformed cells showed phenotypically related alterations. Inter alia, there are specific decreases in the binding of specific lectins (and carbohydrate specific monoclonal antibodies) to senescent IMR-90 cells when compared to Phase II cells (Mann, et al., Mech. Aging Devel. 38, 207-218 (1987)) which are related to the affinity of binding and not to the simple epitope density (Mann, et al., Mech. Ageing Devel. 44, 1-16, (1988); Mann, Intl. Rev. Cytol. 112, 67-96 (1988)). Thus the concept of a three-dimensional oligosaccharide-dependent surface has developed. This surface is functionally related to the traditional glycocalyx by in-plane mobility measurements of the ligand/lectin complex (Mann, et al., Mech. Aging Devel. 38, 219-230 (1987)).
These cell surface display alterations preceded the morphological evidence of senescence. The hypothesis holds that this surface acts as an information filter, accepting and integrating information from the environment, inducing and regulating the subsequent phenotypic change. Thus, the cell surface display is squarely at the forefront of mechanistic studies. Senescent cells down-regulate their surface displays in specific ways with a specific carbohydrate-dependent temporal sequence. In general, the calculated value of Gibb's Free Energy (GFE), which is the sum of all the oligosaccharide-dependent binding energy, provided a gross view of the status of the cell surface. The morphologically senescent IMR-90 cell had a GFE of 10 KCal less than the Phase II cells and therefore was considered down-regulated.
It was also observed that the regulatory phenomenon of the cell surface was not absolutely specific to the onset of senescence. Normal Phase II IMR-90 cells undergo similar down-regulation of their surfaces in response to low density and contact-induced growth inhibition (Mann, et al., Mech. Aging Devel. 44, 17-33 (1988)). The cell surface oligosaccharide status appears to be a generic regulatory mechanism which permits the cell to respond to non-optimal growth conditions. There are specific differences observed between the pre-senescence and the growth-regulatory down-regulation of the surfaces which have yet to be fully explored, but in general, the normal growth control and cellular senescence appear to be linked as sub-components of cellular differentiation phenomena. The overall hypothesis, which integrates both models holds that (i) the morphological manifestations of cellular senescence, seen at PDL (population doubling level) 45 with the IMR-90 model are the result of inappropriate information integration caused at the cell surface by inappropriate down-regulation of the oligosaccharide display; (ii) that similar regulatory events occur during normal growth in response to low-density and contact-induced growth inhibition; and (iii) neoplastic, growth transformed cells have aberrant cell surface oligosaccharide display, which prevent the cell from expressing growth regulation under any conditions.
Sachs L., Sci. Am. 254, 40-47 (1986) proposed that each cell produces its own differentiation factors in response to endogenous "growth factors" which function as growth inducers for the cell. As the cells multiply (proliferate) under the influence of the growth factors, concentrations of cell differentiative factor(s) produced by the multiplying cells increase(s) sufficiently to induce cellular differentiation. Sachs concludes that these cell-produced differentiation factors are specialized proteins, probably as numerous as the cell types whose maturation they induce. The publication also reports that compounds other than cell-produced differentiation factors, as well as other influences, may directly or indirectly induce differentiation in normal and genetically defective cells in vivo, such as steroid hormones, X-rays, vitamins, bacterial lipopolysaccharides, cytosine arabinoside, adriamycin, methotrexate, lectins and some phorbol esters. No one compound effectively induced differentiation over the broad range of cell types tested.
Research on agents potentially useful for regulating or modulating cellular activity in vitro or in vivo extending over half a century has identified compounds erratically effective in affecting the differentiation and/or proliferation of a very narrow range of cell types under carefully controlled conditions. However, such research has been of limited focus in view of the complexity of the poorly-understood pathways by which cellular proliferation and differentiation are regulated, the unpredictable point at which these prior art compounds intervene in these pathways, and the consequently substantially random nature of the effects of these compounds on the basic underlying mechanisms controlling differentiation and proliferation.
Therefore, there is a need to identify substances which directly affect, e.g., normalize, the primitive control mechanism(s) regulating differentiation and proliferation in cells, to which cells can be exposed in vivo and in vitro to obtain a normalizing alteration in cellular differentiative and proliferativa activity over a broad range of cell types. In particular, it is desirable to provide substances which can induce self-adjustment of aberrant cellular behavior to return aberrant cells to normal function, wherein reproductive (proliferative) and differentiative cellular activities are integrated and responsive to the requirements of the host organism.